Impairment of FOXM1 expression in mesenchymal cells from patients with myeloid neoplasms, de novo and therapy-related, may compromise their ability to support hematopoiesis

Bone marrow mesenchymal stem cells (BM-MSCs) exhibit multiple abnormalities in myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML), including reduced proliferative and clonogenic capacity, altered morphology, impaired immunoregulatory properties and capacity to support hematopoiesis. Here, we investigated expression of the FOXM1 gene, a transcription factor driving G2/M gene expression, in BM-MSCs isolated from patients with MDS and AML, de novo and therapy-related, compared to BM-MSCs isolated from healthy donors (HD). We observed a statistically significant downregulation of FOXM1 expression in BM-MSCs isolated from MDS and AML patients, as compared to controls. In parallel, expression of FOXM1 mitotic targets (CCNB1, CDC20, PLK1 and NDC80) was suppressed in patients’ BM-MSCs, as compared to HD. No differences in the expression of FOXM1 and its mitotic targets were observed in BM-mononuclear cells from the different sources. From a functional standpoint, silencing of FOXM1 mRNA in healthy MSC induced a significant decrease in the expression of its targets. In this line, healthy MSC silenced for FOXM1 showed an impaired ability to support hematopoiesis in vitro. These findings suggest that deregulation of FOXM1 may be involved in the senescent phenotype observed in MSC derived from myeloid neoplasms.


Results
Phenotype of MSC in myeloid neoplasms. We studied morphologic alterations in MSC isolated and expanded from the BM-MNC of patients with myeloid neoplasms, including AML and MDS, de novo and therapy-related. All samples were studied at the time of initial MN diagnosis. Compared to normal MSC, at the second expansion passage, patients MSC morphology was disrupted with a larger, flattened and disorganized appearance. Figure 1A shows exemplary pictures of MSC from a MDS patient and a control BM. The number of CFU-fibroblasts was also significantly reduced, with no differences between de novo and therapy-related subtypes (Fig. 1B).

FOXM1 expression levels in AML and MDS.
We studied FOXM1 mRNA expression levels in BM-MSC isolated from patients with myeloid neoplasms as compared to healthy donors. We observed a statistically significant downregulation of FOXM1 in MSC from de novo AML and MDS, and therapy-related MN (t-MN), compared to HD (p = 0.0079, p < 0.0001, p = 0.0006 respectively, Fig. 2A). According to BM-blast counts, a trend for FOXM1 downregulation was observed in MDS versus AML samples, both de novo and therapy-related (p = 0.0552 and p = 0.0593, respectively, data not shown). In AML, the lowest FOXM1 levels were observed in therapy-related subtypes (p = 0.0083, Fig. 2A). On the other hand, there were no differences in FOXM1 mRNA expression levels when comparing BM-MNCs from patients and HD, indicating that FOXM1 downregulation is exclusive of the mesenchymal compartment (Fig. 2B).
Given the age difference between patients and controls (median age 70 years vs. 50 years, p < 0.0001), we assessed whether age could be a confounding factor in the analysis of FOXM1 expression. The Spearman's correlation test showed that FOXM1 deregulation was not correlated with age, as showed in Fig. 3A.
To test whether hypermethylation was the cause of FOXM1 downregulation, we treated 5 MDS-MSC samples with 1 µM decitabine (DAC), for 5 days. This treatment was unable to restore normal FOXM1 expression levels ( Fig. 4), suggesting that FOXM1 gene regulation is not dependent on DNA methylation in the MSC context.  www.nature.com/scientificreports/

Expression of FOXM1 mitotic targets.
To study the functional consequences of FOXM1 downregulation, we assessed expression levels of some of its known mitotic targets (CCNB1, PLK1, NDC80, and CDC20) (21). All genes tested were significantly downregulated in MN-MSC compared to HD-MSC, independent of the MN subtype ( Fig. 2A). Levels of FOXM1 and its mitotic targets in MSC were directly correlated (Fig. 3B), and were independent of the proportion of BM-blasts (data not shown). These quantitative analyses demonstrated that important protein players, acting from mitotic entry (CCNB1 and PLK1) to anaphase onset (CDC20 and NDC80), are expressed at significantly lower levels in MSC isolated from patients with MN, as compared to HD-MSC. FOXM1 target gene levels in MNC were on the contrary similar to that of HD from the same patient group (Fig. 2B).
FOXM1 silencing by siRNA. To investigate the functional effects of FOXM1 downregulation, we silenced FOXM1 mRNA, using a pool of 3 siRNA (si-FOXM1 1,2,3 ) directed against different portions of the FOXM1 gene. These tests were performed in BM-MSC from 5 HD, where expression of the FOXM1 gene was measurable. After 48 h of si-FOXM1 1,2,3 transfection, we observed greater than 90% decrease in the expression of FOXM1 mRNA compared to control siRNA (Fig. 5A). FOXM1 silencing was associated with downregulation of all target genes significantly decreased and was more pronounced for PLK1 mRNA (70%, Fig. 5A).  www.nature.com/scientificreports/ Considering the direct modulation of mitotic targets following FOXM1 gene silencing, we tested whether it would interfere with the cell cycle. After 48 h of FOXM1 silencing in HD-MSC, we did not observe differences in the distribution of cells in the different phases of cell cycle (G1, S, G2-M, Fig. 5B-C). In this line, the number of cells after 48 h of culture did not change (data not shown).
The clonogenic capacity of healthy CD34 + cells has been shown to be impaired after co-culture with MSC isolated from patients with MDS 9 . Given that FOXM1 expression is reduced in these cells, we tested whether inhibition of FOXM1 expression could reproduce the "MDS-MSC phenotype" in healthy MSC. To this end, we determined the frequency of myeloid progenitors using colony-forming unit assays from HD-CD34 + cells co-cultured with HD-MSC. Interestingly, CD34 + cells pre-incubated with FOXM1-deficient HD-MSC gave rise to a significantly lower number of colonies, when compared to those cultured with HD-MSC transfected with control siRNA (Fig. 5D). In detail, CFU-GM, but not CFU-E frequency was significantly reduced (median 80 vs.    5E). These data show that FOXM1 reduction is functionally involved in the reduced ability of MDS-MSC to support hematopoiesis.

Discussion
In the present study, we show that FOXM1 is downregulated in BM-MSC isolated from AML and MDS patients, both de novo and therapy-related, as compared to healthy donors. FOXM1 is a member of the forkhead transcription factor family, which plays an important role in regulating the cell cycle 18,19 . In particular, FOXM1 controls mitotic entry through the periodic upregulation of a group of genes, that are maximally expressed during cell progression through late G2 and into M phase 20 . Two of its target genes are CCNB1 and PLK1, and are part of a positive feedback loop that leads to the phosphorylation of FOXM1, and potentiation of its activity 21,22 . This suggests an intricate inter-regulatory relationship between FOXM1 and PLK1, leading to a cell-cycle control switch. Indeed, all 4 FOXM1 mitotic targets (CCNB1, PLK1, CDC20, NDC80) analyzed in the present study, were significantly downregulated in BM-MSC of MN patients. SiRNA experiments showed that this deregulation depends on FOXM1 expression, which particularly affected the PLK1 gene, which was suppressed by 70%. The correlation between FOXM1 and PLK1 expression has also been reported by Zhang et al., in renal cancer cell lines, where PLK1 suppression induced downregulation of FOXM1 expression 23 . In turn, Dibb et al. showed that FOXM1 and PLK1 are overexpressed in patients with gastric adenocarcinomas 24 , in line with our data on myeloid neoplasms.
During recent years, novel functions for FOXM1 have been identified in cancer cells beyond the simple acceleration of G2-M phase progression 18 . This is exemplified by FOXM1 ability to promote nuclear translocation of β-catenin in gliomas, thereby activating a WNT-regulated program 25 . In this line, we previously reported that some signaling pathways involved in multiple MSC properties, including proliferation, differentiation, and  was also shown to activate the Wnt-β-catenin signaling pathways in MLL-rearranged AML, by directly binding and stabilizing the β-catenin protein, thereby preserving leukemic stem cell quiescence and promoting their self-renewal 27 . We confirm that MSC from AML and MDS cells are functionally impaired, as shown by decreased proliferative and clonogenic capacity, and altered morphology, as compared to normal MSC 7-10,12 . MSC from MN present distinct alterations in the expression of essential hematopoiesis-regulating factors such as CXCL12 and Kit-Ligand, which may underlie the deficient ability of MDS/AML-derived MSC to support healthy CD34 + HSPC 9,17 . Moreover, leukemic cells can actively reprogram healthy MSC towards a disrupted phenotype 17 . In this line, our data showed that repression of FOXM1 by siRNA in healthy MSC is able to impair the clonogenic potential of CD34 + progenitor cells, in particular for CFU-GM, mirroring the effects observed when using MDS or AMLderived MSC. In the same line, silencing FOXM1 inhibited the proliferation and colony formation of liver cancer stem cells, and decreased expression of nuclear antigen and Ki-67 proteins 28 .

Conclusions
Our study provides evidence that silencing FOXM1 inhibits stemness of LCSCs, by decreasing the expression of ALDH2, and represses the proliferation, migration, invasion, and tumorigenesis while inducing the apoptosis of LCSCs.
These data show that ematopoietic insufficiency in MDS is at least in part mediated via disturbed MSC functions, and opens up the possibility of targeting specific niche components to restore the correct function of MN MSCs. In this line, several authors showed that treatment of MDS-MSC may reset their normal features. In particular, Wobus et al., treated MDS-MSCs with the TGF-beta pathway inhibitor luspatercept, increasing the clonogenic potential and the migratory capacity of HSPC, both in vitro and in vivo 29 . Similarly, treatment with 5-Azacytidine improved the stemness potential and proliferation capacity of MSCs in MDS 30 , reverting the phenotype to normal in responding patients 31 . In conclusion, the present study demonstrates that impairment of FOXM1 expression in MSC isolated from de novo and therapy-related myeloid neoplasms may underlie their defective capacity to support normal hematopoiesis.

Methods
Patient characteristics. The study population included 40 patients (16 females, 24 males, median age: 70 years, range: 45-89 years) with newly diagnosed myeloid malignancies (de novo AML, n = 10; de novo MDS, n = 15; and therapy-related MN, n = 15). Patients characteristics at diagnosis are illustrated in Table 1. The diagnosis was established according to standard morphologic and immunophenotypic criteria, according to the World Health Organization (WHO) classification 32 . Bone marrow cells harvested from 10 healthy hematopoietic stem cell donors (3 females and 7 males,median age: 50 years, range: 24-64 years) were used as controls. According to the declaration of Helsinki, all patients and controls gave informed consent to the study, which was approved by the Ethical Committee of Tor Vergata University. www.nature.com/scientificreports/ Isolation and expansion of BM-MSCs. Bone marrow mononuclear cells (MNCs) were isolated by Lympholyte-H density gradient separation (Cedarlane, Euroclone, Italy). Ten millions BM-MNCs were seeded and cultured in MesenCult MSC Basal Medium (Stemcell Technologies, Italy) supplied with Mesenchymal Stem Cell Stimulatory Supplements (Stemcell Technologies) and 1% penicillin-streptomycin (Stemcell Technologies), at 37 °C and 5% CO 2 in a humidified atmosphere. The remaining cells were stored, using Trizol reagent (Life Technologies, Italy), for subsequent RNA extraction. After 24 h of incubation, non-adherent cells were removed, and medium was changed twice a week. When adherent MSCs reached 80% of confluence, cells were detached using 0.25% trypsin-1 mmol/L EDTA (Euroclone) and further expanded for a total of five passages (P). Considering the quick exhaustion number of replications observed in culture of MSC isolated from patients with myeloid neoplasm 8  The expression levels of the mRNAs of FOXM1 gene and its mitotic targets CCNB1 (cyclin B1), CDC20 (cell division cycle 20), NDC80 (NDC80 kinetochore complex component) and PLK1 (polo like kinase 1) were analyzed using a semi-quantitative qRT-PCR assay (IQ™ SYBR® Green Supermix, BIO-RAD) in the QuantStudio 1 instrument (ThermoFisher), with GAPDH as the reference gene.
Real-time PCR was performed on RNA extracted from MSCs at the second passage of expansion and on BM-MNCs at the time of diagnosis. Primers used for each amplification reaction are listed in Supplementary  Table 1. A melting curve (62 °C-95 °C) was generated at the end of each run to verify the specificity of the reactions. Expression of genes with a Ct > 35 cycles was considered absent. The gene expression values, specific for each gene, were expressed as 2 −ΔCt , where ΔCt was Ct (test gene) − Ct (reference gene). A difference in gene expression between patients and controls > twofold associated with a p < 0.05 was considered to indicate statistical significance. FOXM1 specific small interfering RNAs. MSC (2 × 10 5 ), isolated from 5 healthy donors, at the fifth passage of in vitro expansion, were seeded in 25 cm 2 surface area flask at the appropriate density (60-80% of confluence). After 16-18 h, they were then transfected with silencer-GAPDH positive control siRNA (cod 4,390,849, Life Technologies, Italy), with silencer-negative control siRNA (cod 4,390,843, Life Technologies, Italy), si-FOXM1-1 vector (cod s5248, Life Technologies, Italy), si-FOXM1-2 vector (cod s5249, Life Technologies, Italy) and si-FOXM1-3 vector (cod s5250, Life Technologies, Italy), using Lipofectamine® RNAiMAX Transfection Reagent (Invitrogen, Waltham, Massachusetts, USA), according to the manufacturer's protocol. A 10 μmol/L siRNA solution was prepared with deionized water. HD-MSC in the logarithmic growth stage were cultured in seven siRNA-containing medium: negative control, GAPDH positive control, FOXM1 siRNA-1, FOXM1 siRNA-2, FOXM1 siRNA-3, pool of 3 FOXM1 siRNA, and blank control (the mock-vehicle group containing only a transfection reagent without siRNA). GAPDH siRNA positive and the negative siRNA controls were used to assess the transfection efficiency.
After 24, 48 and 72 h of transfection, cells in all si-RNA groups were detached using 0.25% trypsin-1 mmol/L EDTA (Euroclone) and stored using Trizol reagent (Life Technologies, Italy), for subsequent RNA extraction. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.